addis ababa university addis ababa institute of technology
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Addis Ababa University
Addis Ababa institute of technology (AAiT)
School of Chemical and Bio Engineering
Characterization and Treatment of Beseka Lake Water
Quality Using Microbes to reduce Total Dissolved Solid
(TDS) and Alkalinity
A Thesis submitted to Addis Ababa Institute of Technology, In Partial
Fulfillment of the requirement of degree of Master of Science in
chemical Engineering (biochemical Engineering Stream)
By: Yasin Ahmed
Thesis Advisor: Dr. Solomon k.
July, 2018
Addis Ababa, Ethiopia
Addis Ababa University
School of Graduate Studies
Addis Ababa Institute of Technology
School of Chemical and Bio engineering
A thesis submitted to the research and Graduate School of Addis Ababa University, Addis Ababa
Institute of Technology, School of Chemical and Bio-Engineering partial fulfillment of the
requirements for the Degree of Master of Science in Chemical Engineering (Biochemical
Engineering Stream)
Approved by the Examining Board:
Chairperson Signature Date
Dr. Solomon Kiros
Advisor Signature Date
Dr. S. Anuradha Jabasingh
_____________________________ _____________________ ________________
Internal Examiner Signature Date
Dr. Shemelis Kebede
_____________________________ _____________________ ________________
External Examiner Signature Date
DECLARATION
I declare that thesis work entitled ―: Characterization and Treatment of Beseka Lake Water
Quality Using Microbes to reduce Total Dissolved Solid (TDS) and Alkalinity submitted for
master degree at Addis Ababa university is my original work and has not previously been
submitted for degree at this or any other university, and that all resources of materials used for this
thesis has been dully acknowledged /referred.
Name: Yasin Ahmed Signature: _________________ Date: _______________
i
ABSTRACT
Water is one of the natural resource that used for irrigation, industrial, sanitation and other purpose
that facilitate human activity. Lake Beseka is volcanically dammed, endorheic lake, which is
located to the East direction, situated around 200 km from Addis Ababa, the capital city of
Ethiopia. The Lake water is saline and alkaline with high concentration of fluoride so that the water
could not be used for drinking and irrigation purpose. Different infrastructures such as a health
center, high school, youth center, offices and others were affected by the lake flooding. So, this
lake needs to be treatment.
In present study, biological neutralization of saline- alkaline beseka lake water in the presence of
glucose (carbohydrate) as carbon source was studied. To do so, determination of physiochemical
characteristics of the lake water, which was pH, EC, TDS, HCO3-, CO3
2-, total alkalinity, chloride
and fluoride were measured and also isolation of bacteria were carried out. In this study Bacillus
alkalophilus bacteria was also used to compare its treatment performance with isolate (T1).
Treatments of lake water sample at different pH, incubation time and inoculum size were
monitored. Maximum removal of total alkalinity and TDS (Total Dissolved Solid) were observed
at pH, incubation time and inoculum size of 9,72hr and 32ml respectively. Which is resulted in
removal of total alkalinity; 774.669mg/l and TDS;1939.92mg/l for isolated (T1) and total
alkalinity;779.631mg/l and TDS;1944.97mg/l for Bacillus alkalophilus from original value. Based
on these findings, isolated (T1) bacteria were shown to be have good performance for biological
treatment of beseka lake.
ii
ACKNOWLEDGMENTS
Above all, all praise to Allah for his blessing and gratitude, the most gracious and the most merciful
to make all those to be the causes for the accomplishment of this study. Secondly, I want to express
my sincere gratitude to my advisor Dr. Solomon Kiros for excellent scientific guidance, support
in fruitful ideas and editing the thesis material. I also appreciate all laboratory staffs of Chemical
and Bio Engineering School of AAiT specially, Mr. Alene Admas and Ms. Hana for their technical
supports and for creating an excellent and friendly atmosphere in the laboratory. I don’t want also
to forget my friends Meron Asteraye, Mohammed Seid, Tesfaye Kassaw and Yigezu Mekonen for
their constructive suggestions and encouragements during my studies.
I would like to acknowledge Ethiopian biodiversity institute for providing me Bacillus
alkalophilus bacteria strain. I would like to thank jimma university for giving me the chance to
attend my MSc. Program. I am also grateful for the Metahara administrator and Metahara urban
drinking and clear water manager & lab assistant Mr. Muleken which are gave me supportive
documents and the chance to use laboratory facility. It is my greatest pleasure to thank Ato Abera
Degefa, Metahara Research and Development Center Manager for his hospitality and arrangement
of laboratory technicians for my work. I don’t want to forget supervisor Fisseha T. and lab
technician Tolessa Ejeta at Metahara Research and Development Center for their endless supports
throughout my experiments at Metahara.
Last but not least: my heartfelt gratefulness goes to my mother, Rahima Yasin and my father
Ahmed Waday who dedicated their life for my success, and my brother Abdulrazak Ahmed for his
initiation and giving me supportive idea during my work. I want to say thank you for the rest of
my families and relatives. It is your greatest encouragement and sacrifices that brought this work
to a successful end.
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TABLE OF CONTENTS
ABSTRACT ................................................................................................................................................... i
ACKNOWLEDGMENTS ............................................................................................................................ ii
LIST OF TABLES ....................................................................................................................................... vi
LIST OF FIGURES .................................................................................................................................... vii
ACRONYMS ............................................................................................................................................. viii
1. INTRODUCTION .................................................................................................................................... 1
1.1 Background ......................................................................................................................................... 1
1.2 Statement of the Problem .................................................................................................................... 3
1.3.1 Main Objectives ........................................................................................................................... 5
1.3.2 Specific Objectives ...................................................................................................................... 5
1.4 Significance of the study ..................................................................................................................... 5
1.5 Scope of the Study .............................................................................................................................. 5
2. LITERATURE REVIEW ......................................................................................................................... 6
2.1 Brief Description of Lake Beseka and related lakes in Ethiopia......................................................... 6
2.2 Source of saline- alkaline of water ...................................................................................................... 8
2.3 Salinity and alkalinity hazards on human, plants and crops ............................................................... 8
2.4 Remediation measurement taken on Lake Beseka .............................................................................. 9
2.5 Desalination technologies ................................................................................................................... 9
2.5.1 Thermal processes ........................................................................................................................ 9
2.5.2 Membrane processes .................................................................................................................. 11
2.6 Effects of desalination technology .................................................................................................... 12
2.7 Other desalination processes ............................................................................................................. 12
2.8 Chemical ways for neutralization of alkaline polluted water ............................................................ 13
2.8.1 Neutralization with Sulfuric Acid .............................................................................................. 13
2.8.2 Neutralization with Hydrochloric .............................................................................................. 14
2.8.3 Neutralization with Nitric Acid .................................................................................................. 14
2.8.4 Neutralization with Carbon Dioxide .......................................................................................... 14
2.8.5 Neutralization with Phosphoric Acid ......................................................................................... 15
2.8.6 Neutralization with flue gas ....................................................................................................... 15
iv
2.9 Halophilic and alkalophilic Microorganisms .................................................................................... 15
2.10 Alkalophilus reaction mechanism ................................................................................................... 16
2.11 Biological Neutralization of alkaline polluted water ...................................................................... 17
2.11.1 Neutralization of alkaline industrial wastewaters using Exiguobacterium sp .......................... 17
2.11.2 Biotechnological process for neutralizing alkaline beverage industrial wastewater ................ 18
2.11.3 Biological neutralization of highly alkaline textile industrial wastewater ............................... 18
2.11.4 Biological neutralization of chlor-alkali industry wastewater ................................................. 18
2.11.5 Neutralization of alkaline wastewater from pulp and paper industry by Alkaliphiles ............. 19
2.11.6 Neutralization of Alkaline Waste-Waters using a Blend of Microorganisms .......................... 19
3 MATERIALS AND METHODS ............................................................................................................. 20
3.2 Methods............................................................................................................................................. 20
3.2.1 Water Sampling method............................................................................................................. 20
3.2.2 Sample Preparation and analysis ................................................................................................ 20
3.3 Media Composition and Isolation of Bacteria .................................................................................. 22
3.3.1 Media Composition .................................................................................................................... 22
3.3.2 Isolation of Bacteria ................................................................................................................... 23
3.4 Experimental procedures .................................................................................................................. 23
3.4.1 Inoculum preparation ................................................................................................................. 23
3.4.2 Viable Cell Count....................................................................................................................... 23
3.5 Beseka lake water treatment procedures ........................................................................................... 24
3.6 Experimental design for Beseka lake water treatment ...................................................................... 24
3.7 Data analysis ..................................................................................................................................... 25
4 RESULTS AND DISCUSSION .............................................................................................................. 26
4.1 Chemical compositions of Lake Beseka water ................................................................................. 26
4.1.1 pH ............................................................................................................................................... 26
4.1.2 Electrical conductivity (EC) ....................................................................................................... 27
4.1.3 Total dissolved solid (TDS) ....................................................................................................... 27
4.1.4 Chloride (Cl-) ............................................................................................................................. 28
4.1.5 Fluoride (F-) ............................................................................................................................... 28
4.1.6 Alkalinity ................................................................................................................................... 29
v
4.2 Isolation and viable cell count .......................................................................................................... 29
4.2.1 Isolation and selection of bacteria .............................................................................................. 29
4.2.2 Viable cells count for isolate (T1) and Bacillus alkalophilus ..................................................... 29
4.3 Statistical analysis of the experimental result ................................................................................... 30
4.3.1 Statistical analysis for isolate (T1) .............................................................................................. 30
4.3.2 Effect of Process Parameters on the treatments of beseka lake water sample ........................... 34
4.4 Optimization of process factors and response variables for isolate .................................................. 38
4. 5 Comparison of alkalinity and TDS removal from wastewater ......................................................... 39
5. CONCLUSION AND RECOMMENDATION ...................................................................................... 40
5.1 Conclusion ........................................................................................................................................ 40
5.2 Recommendation .............................................................................................................................. 41
REFERENCES ........................................................................................................................................... 42
APPENDIX ................................................................................................................................................. 47
Appendix A: Experimental Results for the isolated and bacillus alkalophilus bacteria ......................... 47
APPENDIX B: 3D plots for the responses: for isolated and bacillus alkalophilus bacteria ................... 49
APPENDIX C: Figures Taken during Laboratory Work ........................................................................ 53
vi
LIST OF TABLES
Table 3.1: media composition ....................................................................................................... 22
Table 3.2: Independent Variables and Levels Used in the BBD for the Beseka Lake water
treatment ................................................................................................................................. 25
Table 4.1 characteristics of Lake Beseka water ............................................................................ 26
Table 4.2 Populations of isolate (T1) and Bacillus alkalophilus ................................................... 30
Table 4.3 Analysis of variance for Total alkalinity ...................................................................... 31
Table 4.4 Analysis of variance for TDS ....................................................................................... 32
Table 4.5 Model adequacy measures for total alkalinity .............................................................. 33
Table 4.6 Model adequacy measures for TDS .............................................................................. 33
Table 4-7: Constrains applied for optimization ............................................................................ 38
vii
LIST OF FIGURES
Figure 1.1 Geographical location of Lake Beseka .......................................................................... 7
Figure 4.1 Effect of pH on (a)Total alkalinity (b) TDS ................................................................ 34
Figure 4.2 Effect of incubation time on (a)Total alkalinity (b) TDS ............................................ 35
Figure 4.3 Effect of inoculum size on (a)Total alkalinity (b) TDS .............................................. 36
Figure 4-4 Interaction effects of pH and inoculum size on reduction of (a) total alkalinity (b)
TDS ......................................................................................................................................... 37
Figure 4-5: Ramp display of desirability ...................................................................................... 39
viii
ACRONYMS
ANOVA Analysis of Variance
ASTM American society for testing and materials
APHA American Public Health Association
BBD Box Behnken Design
CFU Colony Forming Unit
ED Electrodialysis
EPA Environmental Public Agency
EC Electrical Conductivity
EBI Ethiopian Biodiversity Institute
ETB Ethiopian Birr
EEPA Ethiopian Environmental Protection Authority
FAO Food and Agriculture Organization
ISE Ion Selective Electrode
MER Main Ethiopian Rift
MSE Metahara Sugar Estate
MEB Multi-Effect Boiling
MSF Multi- Stage Flash
NE North East
RO Reverse Osmosis
SW South West
TFE Tetrafluoroethylene
TDS Total Dissolved Solid
TBT Top Brine Temperature
VC Vapor Compressor
WWDSE Water Work Design and Supervision Enterprise
1
1. INTRODUCTION
1.1 Background
Water is one of the natural resource that used for irrigation, industrial, sanitation and other purpose
that facilitate human activity. This resource directly or indirectly affected by human activates.
Now day, most of our water bodies are seriously polluted due to rapid population growth, industrial
proliferation, urbanizations, increasing living standards and wide spheres of human activities.
Ethiopia is one of African country rich in ground and surface water that used for different purpose.
However, some of these water bodies polluted naturally due to its saline property like Lake Beseka
and other polluted by human activities.
East Africa is a region of extremely complex meteorological and climatologically phenomena and
one of the most extensive rift systems on earth. The topography of the rift exerts a strong influence
on microclimate, drainage systems, and local ecosystems (Gissila et al., 2004; Grant, 2006). The
Ethiopian rift valley is part of the East African rift system, which extends from the Kenyan border
up to the Red Sea and contains lakes of different hydrological and morphometric characteristics.
One of the lakes in the rift valley which are situated in the awash basin is Lake Beseka, which is
located in the northern Main Ethiopian Rift. Lake Beseka is volcanically dammed, endorheic lake,
which is located to the East direction, situated around 200 km from Addis Ababa, the capital city
of Ethiopia. The lake has become habitat of a variety of birds, fishes, crocodiles and other aquatic
species and plays an important role in the wildlife ecology, as it is located in the northern part of
Awash National Park.
Recent news and discussions organized by Ministry of Water, Irrigation and Energy indicated that
the Lake has created several problems instead of giving advantage to the community. The lake is
expanding as opposed to the other rift valley lakes in Ethiopia, which are shrinking (Alemayehu
et al., 2006). Studies done by Gulilat (2000) and Alemayehu et al. (2006) indicated that the total
surface area of the lake is estimated to be 42 km2, which was about 3 km2 in the 1960’s (Gulilat,
2000; Alemayehu et al., 2006). According to Simon and Michele (2007), the Beseka Lake depth
has increased by >3 m from 1976 to 1997, and continues to rise 15 cm/year, requiring Ethiopia’s
main highway and only railroad to the Gulf of Aden to be raised on an embankment. Additionally,
the lake water cannot be used for irrigation and drinking purpose due to its high salinity, alkalinity
and mineral contents (Olumana and Loiskandl, 2015).
2
Several studies have done to investigate the potential contributors to lake level change and
chemical composition, and several reasons were highlighted such as due to an increase in
groundwater level resulting from excessive irrigation downstream (Zemedagegnehu et al., 1999),
increased recharge from irrigation runoff and Koka Dam construction (Ayenew, 2004), flow of
tectonic spring (hot and cold spring) from ground water (Berihu, 2007), and due to increase in
recharge from submerged hot springs (Anna et al., 2008). All the aforementioned studies agreed
and indicated that the volume and the height level of the lake are drastically increased; as a result,
the lake had created several problems in the surrounding area and is a severe threat to the wellbeing
of the indigenous people and the economic welfare of the nation. However, most of the studies
conducted in the area concentrated on the geological evolution, hydrological and identifying the
cause of lake expansion and assessing the resulting damage. Yet, none of these studies adequately
address treatment methods on the water and its watershed, and techniques in the utilization of the
lake water so as to reduce its expansion, level and volume.
The expansion of Lake Beseka is alarming and has had detrimental effect on the surrounding
biological, physical, hydrological and infra-structural environment. The lake growth has affected
the highway and railway structures which run along its northern shore, and lake has flooded the
highway, one of the import-export lines to the port of Djibouti, several times. The nearby Awash
National Park has been affected indirectly, as displaced nomads encroached into the park, looking
for a settlement area, water and grazing land. The expanding lake is in less than 3 km from the
River Awash, which is the source of drinking water and irrigation for millions of people
downstream. If the lake continues to expand at current rate and other influencing factors remain
the same, the lake will cross the natural water divide and invade the town of Addis Ketema and
join the River Awash. This would be disastrous, as the quality of the river water will be deteriorated
such that Ethiopia’s oldest state-owned sugar factory and agricultural development downstream
would be at risk. Megersa et al. (2009) indicated that the expansion of the lake is affecting both
the groundwater dynamics and soil salinization of the nearby agricultural field and sugarcane
plantation and, if it continuous, the sustainability of the farmlands and plantation itself is under
great risk. Currently, the lake water is saline and alkaline with high concentration of fluoride so
that the water could not be used for drinking and irrigation purpose. Studies showed that the rift
valley region of Ethiopia was plagued by water scarcity and poor water quality (Gizaw, 1996;
Reimnn et al., 2002). These indicated that it is serious threat to the surrounding community health
3
including animal health if they used without treatment and reduce crop productivity and cultivated
crop diversity in the surrounding areas. The aforementioned studies indicated that there was lack
of enough information on the methods to treat the lake.
1.2 Statement of the Problem
Lake Beseka is located south of Fentale Volcano in the main Ethiopian rift. Time to time the
expansion of lake becomes the big challenges for Metahara town and Metahara sugar state in socio-
economic and environmental. According to Megersa (2017b) studies, the expansion of the lake
has already started affecting Metahara Sugar Estate (MSE) in terms of groundwater dynamics, soil
salinization, yield decline, etc. and alarmed us before it brings total devastation.
The extension of Lake Beseka resulted salinization of Abadir farm land and forced the government
to change the affected routes (road and railway) and if it is not take any remedial measures still
threaten routes (road and railway) from Addis Ababa to Djibouti. The lake flooded fully and
partially about 150 ha of the sugar cane plantation and over 2000ha of grazing land. The Lake
level rise and flooding created problems on the factory activities. Recent data done by WWDSE
in November 2010 shows that flooded and damaged area from sugar factory estimated to be 178.4
hectares (Dershaye, 2017).
Different infrastructures such as a health center, high school, youth center, offices and others were
affected by the lake flooding. From these, more than 3,500ha of valuable rural farmlands, about
36.45km2 valuable woodlands were lost by over flooded during the last three decades. As a result
of Lake Beseka level rise and expansion, 5 households with about 25 members were completely
displaced and 9 households in Kebele 01 of Metahara town administration were slightly affected.
According to the Metahara town land administration office, at present time totally 422 households
were displaced due to the lake water over flooding.
The Metahara town administration has faced serious challenges on investment attraction due to the
beseka lake expansion and flooding that discouraging the investment activities of the town and
different social services. According to the administration of Metahara town, the socioeconomic
and development activities of the town was severely affected and depressed due to expansion and
flooding of the lake which affect the social and companies invested in and around the town. For
instance, 122.68 hectares of investment areas were covered by the lake water, as well as Dandi
4
Gudina tumsa foundation (Gudina Tumsa Foundation Office, student’s dormitory and high school)
and public health center with surface area of 20ha and 15ha respectively were lost due to unlimited
expansion and flooding of beseka lake. Moreover, companies that have invested earlier in and
around Metahara town such as Anino Trading, Mofti Nuredin, Saf Trading, animal fattening and
Adey Abeba textile have left the area, which can be good sources of job opportunity to the town
community, and created negative image on other investors (Dershaye, 2017).
The lake level rise and expansion also created health impacts on communities. It can aggravate
bone and teeth decay problems. It also aggravates problems on safe water provision as the saline
lake water imposes pressure on water treatment. The lake has a potential to join Awash River in
the near future, thereby impacting all downstream irrigation developments in the Awash Basin and
affecting the livelihood of the people depending on the water resources of the basin (Megersa,
2017a). Currently, daily the communities shocked since they are assuming that one day the town
submerged by Lake Besaka water.
To overcome the problem of the lake, the former Ministry of Water and Energy of Ethiopia spent
35 million ETB to make drainage lake water into Awash River (2% of Awash river water by
volume) starting from 2004(Abate et al, 2016). However, the draining of Beseka lake water into
the Awash River might be causes the deposition of salinity and other minerals within soil of the
middle Awash farm through the time. After long period of time, in order to leach the deposited
minerals from the affected soil take another cost. Considering the fact that up to now only limited
research has been performed by various organizations, a wide knowledge gap exists regarding the
mechanisms to reduce the expansion through biological treatment by utilizing appropriate
microbial species so that the lake water can be used for construction, watering of tree seedlings
and irrigation.
5
1.3.1 Main Objectives
The main objective of the present research is characterization and treatment of Beseka Lake
water quality using microbes to reduce Total Dissolved Solid (TDS) and Alkalinity
1.3.2 Specific Objectives
• To characterize the physic-chemical composition of Lake Beseka water.
• To isolate bacteria from Beseka lake.
• To treat the lake water using microbes at different concentrations, pH and incubation time.
• To optimize the biological treatment process for reduction of salinity (TDS) and alkalinity.
1.4 Significance of the study
Water plays a significant role in every organism on earth. Today, this natural resource polluted
due to nature of its property, industrial plants, chemicals, household activities and other uses of
water in the community.
It is known that, Lake Beseka cover large surface area in Metahara town and becoming a challenge
for Metahara community and Metahara sugar factory because of expansion of lake level and not
has any function due to saline property of lake. Up to now, no visible treatment means applied
rather than diversion of it to Awash River. Since the biological treatment employed in this study
is natural treatment process, it is economic and environmental friendly alternatives to chemical
treatment methods. The study can also provide information about natural resource management
through water treatment.
1.5 Scope of the Study
To achieve the objectives of the research study, biological treatment of Beseka Lake water
experiments was carried out with generate series of lab work by using Design of Experiment
software. This thesis generally covers isolation of bacteria from Beseka Lake and uses it for
treatment purpose and investigation of the effects of pH, incubation time and dosage of inoculum
during treatment. And evaluate the treatment performance of both the isolated bacteria and,
provided bacteria from Ethiopian biodiversity institute.
6
2. LITERATURE REVIEW
2.1 Brief Description of Lake Beseka and related lakes in Ethiopia
Ethiopia is gifted with many Lakes and rivers that comprise diverse aquatic ecosystems of great
scientific interest and economic importance. The Ethiopian Rift Valley, as part of the Great East
African Rift Valley, splits the Ethiopian highlands into northern and southern halves, forming a
floor in between occupied with the chain of lakes characterized by varied size, hydrological and
hydrogeological settings. With the total area of 1.3 million ha, it encompasses three major water
basins from NE to SW. The Awash basin with Koka, Beseka, Gemari and Abe, the Central
Ethiopian Rift Valley with Lake Ziway, Langano, Abiyata and Shalla and the Southern basin with
Awassa, Abaya, Chamo and Chew-Bahir as the most important lakes (Tigist, 2009).
Lake Beseka is one of the Awash Basin situated Lake, located in the Fentalle Woreda, East Showa
Zone of Oromia regional state at about 200 km southeast of Addis Ababa, the capital city of
Ethiopia. It is volcanically dammed, endorheic lake located in the northern part of the Main
Ethiopian Rift (MER). It situated on the main road from Addis Ababa to the port of Djibouti. It is
located at the southern end of the Awash National Park, one of Ethiopia’s major parks. In contrast
to East African lakes, the size of Lake Beseka has increased dramatically since 1960. The total
surface water catchment of the lake is about 500 km2 (Olumana, 2017a, 2017b). The total surface
area of the lake is estimated to be about 50 km2 in the year 2015, which were about 3 km2 in the
1960’s. As the area is situated in the upper most part of MER, Lake Beseka is vulnerable to the
occurrences of different tectonic and volcanic activities. It has saline-alkaline properties with high
fluoride contents. The lake expansion affects the surface- and ground-water dynamics and soil
properties of the region and the condition is specifically dangerous for the sustainability of MSE
and Metahara town (Olumana, 2017b). Moreover, Beseka Lake is instilling terror to local and
international investors and also communities due to its expansion and saline -alkaline properties of it.
7
Figure 1.1 Geographical location of Lake Beseka (source: Olumana, 2017a)
Like Lake Beseka, Chew Bahir, Abiyata and Shalla Lakes are found in Ethiopian Rift Valley Lake
and have saline properties. Lake Abiyata is a shallow, highly productive, saline-alkaline lake,
whose muddy shoreline supports one of the most notable collections of bird life (flamingo, white
pelicans, etc.) in Africa. The maximum reduction in the water level of Lake Abiyata occurs at same
time with the period of large-scale water abstraction for soda ash production, and for irrigation
with water from Lake Ziway after the 1980s(Ayenew and Legesse, 2007). Lake shall also have
alkaline nature, which discourages its use for irrigation purpose(Belete et al., 2016).
Chew Bahir is at the southern end of the Ethiopian section of the Great Rift Valley. It lies across
the border of South Omo Zone of Southern Nations, Nationalities and Peoples‟ Region (SNNP) to
the west, and the Borena Zone of Oromia Region to the East. The water of Chew Bahir is so highly
saline that it cannot be used for either irrigation or domestic purposes (Daniel, 2014). Lake Afdera
is another saline lake located in the afar region, Ethiopia. Since the lake is highly saline, only used
for extraction of salt. According to 1988 studies, the pH of the lake is pH 6.55. Unlike the other
8
saline lake in Ethiopia (lakes shala, beseka, chitu, chew Bahir and abijata) the pH is low that is
acidic in nature(Getahun, 2001).
2.2 Source of saline- alkaline of water
Water is one of the most abundant resources on earth, covering three fourths of the planet’s surface.
About 97% of the world’s water resources is in the oceans and seas and is too salty for most
productive uses(Hailu et al., 2016; Méridien, 2013). The sources of such saline and alkaline can
be due to the levels of hard-water minerals and release of basic industrial and agricultural effluents,
dissolution of carbonate rocks, the high anthropogenic activities and geological weathering
conditions(Olumana and Loiskandl, 2015). High saline wastewater is mainly discharged from
industry like chemical, pharmaceutical, petroleum industries, fish caning, seafood processing,
meatpacking and tannery ( Lefebvre and Moletta, 2016; Méridien, 2013),and natural saline lake.
2.3 Salinity and alkalinity hazards on human, plants and crops
Salinity is a measure of the amount of salts in the water. The most important criteria for evaluating
salinity hazards are the total concentration of salts. The quantity of slats dissolved in water is
expressed in terms of Total Dissolved Solids (TDS), mg/l (ppm) (Siosemarde et al., 2010). Mostly
cations like Na+, Ca2+ and Mg2+ and anions like Cl-, SO42-, HCO3
- and CO32- are the major
constituents contained in saline waters.
Salinity and alkalinity hazards on human: Using saline water causes the problems like stomach
and laxative effects. Drinking alkaline water lead to a condition called metabolic alkalosis, which
may cause confusion, nausea, vomiting, hand tremors and so on(Méridien, 2013).,
Salinity and alkalinity hazards on plants and crops: Plants growth is adversely affected with
saline water, primarily through the effectives of excessive salts on osmotic pressure of the soil
solution resulting in reduced availability of water. The first reaction of plants to application of
saline water is reduction in germination (Verma, 2012). Some waters, when used for irrigation of
crops, have a tendency to produce alkalinity hazards depending upon the absolute and relative
concentrations of specific cations and anions. Salinity may cause nutrient deficiencies or
imbalances of the plant, due to the competition of Na+ and Cl– with nutrients such as K+, Ca2+, and
NO3-(Hu & Schmidhalter, 2005). Irrigation with sodic waters contaminated from Na+ relative to
Ca2+ and Mg2+ and high carbonate (CO32-) and bicarbonate (HCO3
-) leads to an increase in
9
alkalinity and sodium saturation in the soil. The increase in exchangeable sodium percentage
adversely affected soil physical properties including infiltration and aeration (Verma, 2012).
2.4 Remediation measurement taken on Lake Beseka
To overcome the beseka lake problems and challenges, government and other stakeholders have
taken limited treatment mechanisms such as diverting 2% of the Beseka lake water to Awash River
in daily basis (Abate et al, 2016). However, such method might have negative effect on the
downstream through the accumulation of salt and fluoride in the long-term period, and so it is
better to search treatment mechanism rather than diverting.
2.5 Desalination technologies
Water is polluted due to municipal, agricultural, industrial or natural activity. The conditions of
the polluted water without treatment will be deleterious to the organism and plants in the river,
lake and human health. Desalination is a separation process that produces two streams, one that
has a low concentration of salt (treated water or product water), and the other with a much higher
concentration than the original feed water(brine). The two major types of technologies that are
used around the world for desalination are thermal and membrane technology. Both technologies
need energy to operate and produce fresh water.
2.5.1 Thermal processes
Thermal technologies, as the name implies, involve the heating of saline water and collecting the
condensed vapor (distillate) to produce pure water. These processes include Multi-Stage Flash
(MSF), Multi-Effect Boiling (MEB), and Vapor Compression (VC) Distillation. In all these
processes, condensing steam is used to supply the latent heat needed to vaporize the water.
A. Multi-stage flash desalination (MSF)
Multi-stage flash desalination (MSF) is a thermal distillation process that involves evaporation and
condensation of water. The evaporation and condensation steps are coupled to each other in several
stages so that the latent heat of evaporation is recovered for reuse by preheating incoming water.
In the so-called brine heater, the incoming feed water is heated to its maximum temperature (top
brine temperature) by condensing saturated steam from the cold end of a steam cycle power plant
or from another heat source. The hot seawater then flows into the first evaporation stage where the
10
pressure is set lower. The sudden introduction of hot water into the chamber with lower pressure
causes it to boil very quickly, almost flashing into steam. The vapor generated by flashing is
condensed on tubes of heat exchangers that run through the upper part of each stage. The tubes are
cooled by the incoming feed water going to the brine heater, thus pre-heating that water and
recovering part of the thermal energy used for evaporation in the first stage(Méridien, 2013). The
top brine temperature (TBT) range is usually within 90 to 120ºC. Although higher efficiency is
observed by increasing TBT beyond 120ºC, scaling and corrosion at high temperature affects the
process significantly. To accelerate flashing in each stage, the pressure is maintained at a lower
value than that in the previous stage. Hence, the entrance of heated seawater into the flash chamber
causes vigorous boiling caused by flashing at low pressure (Bahar et al., 2013). Multi-stage flash
(MSF) units are widely used in the Middle East (particularly in Saudi Arabia, the United Arab
Emirates, and Kuwait) and they account for 34% of the world’s seawater desalination. The MSF
process requires a considerable amount of steam for the evaporation process and also significant
amounts of electricity to pump the large liquid streams(Méridien, 2013).
B. The multiple-effect boiling (MEB)
The MEB process is composed of a number of elements, which are called effects to desalinate
saline water. In this process, the feed water is sprayed or otherwise distributed onto the evaporator
surface (usually tubes) of different chambers (effects) in a thin film to promote evaporation after
it has been preheated in the upper section of each chamber. The evaporator
tubes in the first effect are heated by steam extracted from a power cycle or from a boiler. The
steam from one effect is used as heating fluid in another effect which while condensing, causes
evaporation of a part of the salty solution. The produced steam goes through the following effect,
while condensing; it makes some of the other solution evaporate and so on.
In principle, MEB plants can be configured for high temperature or low temperature operation. At
present, they operate at top brine temperatures below 70ºC to limit scale formation and
corrosion. The top brine temperature can be as low as 55ºC which helps to reduce corrosion and
scaling and allows the use of low-grade waste heat. If MEB coupled to a steam cycle, the power
losses will be much lower than those obtained when coupling a MSF plant , and even standard
condensing turbines may be used instead of back-pressure turbines(Méridien, 2013).
11
C. The vapor-compression (VC)
In a VC plant, heat recovery is based on raising the pressure of the steam from a stage by means
of a compressor. The condensation temperature is thus increased and the steam can be used to
provide energy to the same stage it came from or to other stages. As the conventional MEB system,
the vapor produced in the first stage is used as the heat input to the second effect which is at a
lower temperature. The vapor produced in the last effect is then passed to the vapor compressor
where it is compressed until its saturation temperature is raised before it is returned to the first
effect. The compressor represents the major energy input to the system and since the latent heat is
effectively recycled around the plant.
2.5.2 Membrane processes
Membrane processes include Reverse Osmosis (RO) and Electrodialysis (ED). Whereas ED is
suitable for brackish water, RO can be used for both brackish and seawater.
A. Reverse osmosis (RO)
Osmosis is a natural phenomenon by which water from a low salt concentration passes into a more
concentrated solution through a semi-permeable membrane. When pressure is applied to the
solution with the higher salt concentration solution, the water will flow in a reverse direction
through the semi-permeable membrane, leaving the salt behind. This is known as the Reverse
Osmosis (RO) process. The pressure needed for separation ranges within 50 bars (seawater) to 20
bars (brackish water) (Bahar et al., 2013). RO membranes are sensitive to pH, oxidizers, and a
wide range of organics, algae, bacteria, and deposition of particulates and fouling. Therefore, pre-
treatment of the feed water is an important process step and can have a significant impact on the
cost and energy consumption of RO. Recently, micro-, ultra- and nano-filtration has been proposed
as an alternative to the chemical pre-treatment of raw water in order to avoid contamination of the
seawater by the additives in the surrounding of the plants. RO post-treatment includes removing
dissolved gases (CO2), and stabilizing the pH via the addition of calcium or sodium salts, and the
removal of dangerous substances from the brine.
B. Electrodialysis (ED)
Electrodialysis is an electrochemical process in which the salts pass through the cation and anion
membranes, leaving the water behind. It is a process typically used for brackish water. Because
12
most dissolved salts are ionic (either positively or negatively charged) and the ions are attracted to
electrodes with an opposite electric charge, membranes that allow selective passage of either
positively or negatively charged ions accomplish the desalting(Subashini, 2015)
2.6 Effects of desalination technology
By the year 2025, two out of every three persons in the world could be living in water-stressed
regions. One of the solutions to increase potable water availability is desalination of brackish water
and seawater. It is estimated that worldwide desalination capacity will increase by 2015 to 97.5
million m3of water per day, with energy accounting for 40% of the total water cost. Energy
demands for water desalination range from 650 𝐾𝑊ℎ
𝑚3 for energy intensive single-stage evaporation
of seawater to 68 𝑘𝑊 ℎ
𝑚3 for multistage flash evaporation and 3.7
𝐾𝑊ℎ
𝑚3for reverse osmosis (RO)
(Mehanna et al., 2010). .
Approximately 78% of the desalinated water in Saudi Arabia is produced from seawater, and the
main technologies used by the Saline Water Conversion Corporation (SWCC) are multi-stage flash
distillation (MSF), reverse osmosis (RO), and multi-effect distillation (MED). The unit capital cost
in 2010 for seawater desalination plants in the Arab region has been estimated to be between
$1000_$2000 per m3/day, while energy costs are both highly variable and can represent 50_77%
of the total operating costs ($0.46_$0.60/m3) in cogeneration plants(Kajenthira et al., 2012). The
majorities of existing desalination plants are powered by fossil fuels and result in substantial
greenhouse gas emissions. Desalination also results in the production of concentrated brine which
may affect coastal water quality and marine life due to its discharge temperature and exceedingly
low concentrations of dissolved oxygen if not disposed in good manner(Kajenthira et al., 2012;
Méridien, 2013).
2.7 Other desalination processes
I. Freezing
During the process of freezing, dissolved salts are excluded during the formation of ice crystals.
Under controlled conditions seawater can be desalinated by freezing it to form the ice crystals.
Before the entire mass of water has been frozen, the mixture is usually washed and rinsed to
remove the salts in the remaining water or adhering to the ice.
13
The ice is then melted to produce fresh water. Therefore, the freezing process is made up of cooling
of the seawater feed, partial crystallization of ice, separation of ice from seawater, melting of ice,
refrigeration, and heat rejection. The advantages of freezing include a lower theoretical energy
requirement, minimal potential corrosion, and little scaling or precipitation. The disadvantage of
freezing involves handling ice and water mixtures which are mechanically complicated to move
and process (Khawaji et al., 2008).
II. Solar evaporation
Solar desalination process is similar to a part of the natural hydrologic cycle in which the seawater
is heated by the sun’s rays to produce water vapor. The water vapor is then condensed on a cool
surface, and the condensate collected as product water. An example of this type of process is the
green house solar still, in which the saline water is heated in a basin on the floor and the water
vapor condenses on the sloping glass roof that covers the basin.
III. Using microbial desalination cells to reduce water salinity prior to reverse osmosis
A new concept for water desalination was recently demonstrated based on using electrical power
generated by bacteria in devices called microbial desalination cells (MDCs). The concept is similar
to water Electrodialysis in the fact that there is a voltage applied between the chambers in order to
drive the ions out of the desalination compartment, but instead of using an external source of
electrical energy desalination is achieved with the current and potential generated by bacteria in
the MDC. When a lower salinity wastewater stream is used in the anode chamber to desalinate the
water in the middle chamber, the MDC also has characteristics of a reverse Electrodialysis (RED)
process as the salinity gradient between the two chambers can contribute to current
generation(Mehanna et al., 2010).
2.8 Chemical ways for neutralization of alkaline polluted water
2.8.1 Neutralization with Sulfuric Acid
Sulfuric Acid (H2SO4) is the most widely used and produced chemical in the world. Available in
concentrations ranging from 0% to 98%, sulfuric acid is most economical of all and used
universally for neutralization reactions. It is easier and safer to use than HCl or HNO3 and is more
potent than all of the other acids except for phosphoric acid. Sulfuric acid is typically used in
concentrations ranging from 25% to 96%. However, 30% to 50% concentrations of Sulfuric acid
14
are generally recommended (Kumar et al., 2007). Reactions times of 15-30 minutes are needed.
The formation of calcium sulfate is possible when calcium containing polluted waters is
neutralized (Eckenfelder et al., 1989).
2.8.2 Neutralization with Hydrochloric
Hydrochloric Acid (HCl), also known as muriatic acid, is the second most commonly used acid in
industry, Sulfuric acid being the primary choice since it is more effective and relatively
inexpensive. Reactions times of 15-20 minutes are needed. At a maximum available concentration
of 37%, HCl is about 1/3 as potent as Sulfuric acid, thus making it relatively more expensive to
use. Depending on temperature and agitation, HCl at concentrations above 10% evolves hydrogen
chloride vapors which combine with the water vapors present in the air. The gas thus formed, is
highly corrosive and attacks all metallic objects including building structures, sprinkler heads,
copper wiring, stainless steel, etc. Therefore, it must be properly vented or used outdoors where
the gasses can easily dissipate (Eckenfelder et al., 1989; Kumar et al., 2007).
2.8.3 Neutralization with Nitric Acid
Nitric Acid (HNO3) though a widely used chemical in many industries it does not enjoy the
popularity of hydrochloric or Sulfuric acid, as it is more expensive to use than either of them.
Nitric acid evolves noxious gas which on combines with water vapors present in the air. The gas
is highly corrosive and attacks all metallic objects including building structures, sprinkler heads,
copper wiring, stainless steel, etc. Therefore, it must be properly vented or used outdoors where
the gasses can easily dissipate (Kumar et al., 2007).
2.8.4 Neutralization with Carbon Dioxide
Carbon Dioxide (CO2), the third most concentrated gas found in earth's atmosphere, CO2 is itself
not an acid. It forms carbonic acid (H2CO3) when dissolved in water; and it is this carbonic acid
that brings about the neutralization of alkalinity in Solution. The most appealing feature of CO2 is
that it will not lower the pH of water below 7.0 for all practical purposes. Additionally, CO2 is
non-corrosive. However, as it is heavier than air asphyxiation is a hazard. Carbon dioxide is
difficult to use and its use is limited because the gas must be dissolved into Solution to be used.
This requires the use of a carbonator, or some method to dissolve the gas into solution. Significant
out-gassing also occurs, which does not hold a problem unless the process also requires the settling
of Solids. In cement pouring operations large amounts of alkaline wastewaters are generated. It is
15
an excellent choice for Such applications as the site is temporary, the gas is nonhazardous, can be
used in-line assuming retention and mixing is considered and is self-buffering so regardless of
dosage it will not lower the pH below 7.5-7.0 (Verma, 2012).
2.8.5 Neutralization with Phosphoric Acid
Phosphoric Acid (H3PO4), very widely used in the production of agricultural fertilizers and
detergent products it is a relatively inexpensive acid. However, it still does not compete well with
sulfuric and hydrochloric acid as it is a weak acid and does not fully disassociate in water at normal
concentrations. This renders it safer to use compared to sulfuric or hydrochloric acid and the
evolution of gasses is rare. It tends to buffer neutralization reactions and this makes for a slower
reaction that is easier to control. Due to its cost (as compared to Sulfuric) and availability,
phosphoric acid is not commonly used in neutralization system (Kumar et al., 2007)
2.8.6 Neutralization with flue gas
Flue gas also contains a considerable amount of carbonic acid (7.5 to 12%), which can be used for
neutralization of alkaline wastewaters. It is produced in heating plants and power stations and is
therefore available free of charge. Neutralization with flue gas requires a large amount of flue gas;
this gas is not injected into the air suction pipe, but distributed over the pipe cross section via a
branch line. This means that only flue gas or only air can be injected. The injection is controlled
by a multi way a valve. If the pH value exceeds the legally admissible discharges limits, a pH
control and measuring devices activates the multi way valve and the flue gas injection starts
(Verma, 2012).
2.9 Halophilic and alkalophilic Microorganisms
Halophilic microorganisms comprise a heterogeneous group of microorganisms and require salts
for optimal growth. They have been isolated from diverse salinity environments, varying from
natural brines, hyper saline lakes to saturation salinities. Microorganisms able to grow in the
presence of salt are found in all three domains of life: Archaea, Bacteria, and Eukarya(Margesin
and Schinner, 2001; Waditee-sirisattha et al, 2016). Non-halophilic microorganisms, able to grow
in the absence as well as in the presence of salt, are designated halotolerant; those halotolerants
that are able to grow above approximately 15% (w/v) NaCl (2.5 M) are considered extremely
halotolerant. Microorganisms requiring salt for growth are referred to as halophiles. Halophiles
16
can be distinguished as Slight halophiles (0.2-0.5 M NaCl), moderate halophiles (0.5-2.5M NaCl)
and extreme halophiles (2.5-5.5M NaCl)(Margesin and Schinner, 2001; Zhuang et al, 2010).
The term “alkaliphile” is used for microorganisms that grow optimally or very well at pH values
above 9 but cannot grow or grow only slowly at the near-neutral pH value of 6.5. Alkaliphiles
include prokaryotes, eukaryotes, and archaea. The cell surface may play a key role in keeping the
intracellular pH value in the range between 7 and 8.5, allowing alkaliphiles to thrive in alkaline
environments. Alkaliphiles can be classified as obligatory and facultatively alkaliphilic organisms.
Obligatory alkaliphilic microorganisms cannot grow below pH 9. Facultatively alkaliphilic
organisms show optimal growth at pH 10.0 or above but they also have the ability to grow at
neutral pH(Coban, 2004). Alkaliphiles consist of two main physiological groups of
microorganisms; alkaliphiles and haloalkaliphiles. Alkaliphiles require an alkaline pH of 9 or more
for their growth and have an optimal growth pH of around 10, whereas haloalkaliphiles require
both an alkaline pH (>pH 9) and high salinity(Mudidi, 2014).
2.10 Alkalophilus reaction mechanism
In order to survive at alkaline environment and high salt concentrations, alkalophilic
microorganisms have to maintain an osmotic balance with their external environment. The
following point shows the withstanding ability of the alkalophilic microorganisms at alkaline
environment and high salt concentrations:
Internal pH: The cell surface is a key feature in maintaining the intracellular neutral environment
separate from the extracellular alkaline environment. Alkaliphiles use proton pumps to maintain a
neutral pH internally, and so the intracellular enzymes from these microorganisms need not to be
adapted to extreme growth conditions. The Na+/H+ antiporter protein present on the plasma
membrane enables cells to adapt to a sudden upward shift in pH and to maintain a cytoplasmic pH
that is 2 to 2.3 units below the external pH in the most alkaline range of pH for growth. These
mechanisms of these membrane proteins play a key role in keeping the intracellular pH value in
the range between 7 and 8.5(Mudidi, 2014).
Cell Walls: Alkalophiles have a peptidoglycan cell wall, although in addition to this they have
negatively charged cell wall polymers. Alkaliphilic Bacillus species contain certain acidic
polymers, such as galacturonic acid, gluconic acid, glutamic acid, aspartic acid and phosphoric
acid. These function as a negatively charged matrix and reduce the pH values at the cell surface
17
because the cell membrane is very unstable at alkaline pH values below the optimum pH for growth
and therefore must be kept below 9. Therefore, it helps to stabilize the cell membrane(Horikoshi,
1999). They may give the cell its ability to adsorb sodium and hydronium ions and repulse
hydroxide ions and, therefore, may assist cells to grow in alkaline environments.
Na+ ions and membrane transport: Alkaliphiles require Na+ ions for growth and they have
exhibited vigorous growth at pH range of pH 9 to 11. In Na+ ion–dependent transport systems, the
H+ ion is exchanged with Na+ by Na+/H+ antiporter systems, thus generating a sodium motive
force, which drives substrates accompanied by Na+ into the cells. Incorporation of substrates at pH
9 is observed to greatly increase and the presence of Na+ significantly enhances the incorporation.
Plasma membranes play a role in maintaining pH homeostasis by using Na+/H+ antiporter systems,
K+/H+ antiporter and ATPase drives H+ expulsion(Mudidi, 2014).
2.11 Biological Neutralization of alkaline polluted water
Compare to chemical methods, biological treatment method is a cost effective, sustainable,
nontoxic downstream process, and environmental friendly methods.
2.11.1 Neutralization of alkaline industrial wastewaters using Exiguobacterium sp
A facultatively alkaliphilic bacterium capable of lowering the pH of highly alkaline wastewater
from 12 to 7.5 within just 2hr.The bacterium was isolated from industrial wastewater drained
sludge of a beverage industry. The metabolic product which the bacterium produces in order to
neutralize alkaline wastewater was found to be carboxylic acid in nature by Fourier Transfer-
Infrared (FT-IR) spectroscopy. The isolate belongs to genus Exiguobacterium as determined by
16S rRNA gene sequencing and biochemical characterization. It was found to be gram positive,
no spore-forming, nonmotile, rod shaped, growing aerobically and anaerobically at a wide range
of temperatures (5-40ºC), pH (6.5-12.0) and NaCl concentrations (0-9. 5%).The strain was able to
hydrolyze starch and was catalase positive but lacked oxidase or amino peptidase activity. Acid
was produced from glycerol, cellobiose, D-mannose, Mannitol, methyl α –D-glucoside, amygladin
and arbutin. From the 16S rRNA gene sequence analysis, the strain appeared to be most similar
(99%) to the sequence from Exiguobacterium aurantiacum strain DSM6208. Neutralization of
highly alkaline waste water without addition of any external carbon source highlights the potential
use of isolate as an alternative to the conventional acid neutralization method for treatment of
alkaline wastewater (Verma, 2012).
18
2.11.2 Biotechnological process for neutralizing alkaline beverage industrial wastewater
A biotechnologically pure culture of the strain Exiguobacterium sp., identification number MTCC
5183, said strain being capable of growth in a medium with a pH in the range of 10-12 and being
capable of lowering a pH of 12 to 11.5 of beverage industrial waste water to a neutral pH of 7.5
to 7.00 within a period of 1-1.5 hours, said strain being gram positive, non-motile, rod shaped and
oxidase negative, and being capable of hydrolyzing starch and producing acids from glycerol,
cellobiose ,D-mannose, Mannitol, methyl α-D-glucoside, amygdalin and arbutin (Kumar et al.,
2008).
2.11.3 Biological neutralization of highly alkaline textile industrial wastewater
Bacterium, Kurthia sp., is aerobic in nature, is gram positive, shows optimal growth at a
temperature in the range of 25-42ºC., is capable of growth in a high pH environment (pH= 12), is
capable of hydrolyzing starch, is motile, catalase positive and reduces nitrate. The bacterium
Kurthia sp., as screened above, was inoculated in 200-220ml basal carbonate medium. The culture
was incubated at 35-37ºC for 8 hours under shaking conditions (100-120 rmp). After observing
the heavy bacterial growth (optical density (O. D=1.5)), the culture was centrifuged at 5,000-7000
rmp at about 1-4ᴼC. The culture pellet was added in a flask containing 200ml textile industrial
waste water (pH 12-11.5). The bacterial pellet is used in a ratio in the range of 1:5 to1:10 to the
wastewater. The flask was kept at shaking conditions (100-120 rmp). Decrease in pH was been
capable to bring the pH from 12- 11.5 to 7.5-7.0 with a short period of time 1.5-2 hours, and pH
was monitored by a pH meter. (Kumar et al., 2007).
2.11.4 Biological neutralization of chlor-alkali industry wastewater
The present work reports biological neutralization of chlor-alkali industrial effluent by an
alkaliphilic bacterium, isolated from the Gujarat coast, which was identified as Enterococcus
faecium strain R-5 on the basis of morphological, biochemical and partial 16S rRNA gene
sequencing. The isolate was capable of bringing down the pH of waste water from 12 to 7.0 within
3h in the presence of carbon and nitrogen sources, with simultaneous reduction in total dissolved
solutes (TDS) up to 19-22%. This bacterium produces carboxylic acid, as revealed by FT-IR
analysis, which facilitated neutralization of alkaline effluent. The presence of unconventional raw
materials viz. Madhuca indica flowers or sugar cane bagasse as carbon and nitrogen sources could
effectively neutralize alkaline effluent and thus making the bioremediation process economically
19
viable. The time required for neutralization decreased with an increase in inoculum size (from 5ml
to 20ml of bacterial suspension with specified ceil density per 100 ml of medium) inoculated in
200ml of chlor-alkali industry wastewater supplemented with carbon and nitrogen sources. This is
first report on biological neutralization of a chlor-alkali industrial effluent (Jain et al., 2011).
2.11.5 Neutralization of alkaline wastewater from pulp and paper industry by Alkaliphiles
Two alkaliphilic bacterial strains T1 and T2 capable of lowering pH and alkalinity of alkaline
wastewater from pulp and paper industry were isolated. Bacterial strain T1 reduces the pH from 12
to 8.1 and alkalinity from 1040mg/l to 480mg/l whereas strain T2 reduces the pH from 12 to 7.95
and alkalinity from1080mg/l to 460mg/l. Both alkaliphilic bacterial strains were gram positive and
spore forming. Strain T1 has regular margin, glistering, convex and oval round in shape whereas
strain T2 has regular margin, flat and irregular in shape. Both the strains can grow at wide range
of temperatures (10-44ºC), pH (6-12), and NaCl concentrations (0-20%). Strains T1 and T2 can
utilize all the different types of carbohydrates. Strain T1 can hydrolyze starch and show positive
results for lipase screening, pectinase screening and gelatin liquification test. Neutralization of
highly alkaline wastewater without addition of any external carbon source highlights the potential
use of the isolate as an alternative to the conventional acid neutralization method for treatment of
alkaline wastewater (Verma, 2012).
2.11.6 Neutralization of Alkaline Waste-Waters using a Blend of Microorganisms
The pre-treatment of alkaline waste-waters, generated from various industries like textile, paper &
pulp, potato-processing industries, etc., having a pH of 10 or higher, is essential. The pre-treatment,
i.e., neutralization of such alkaline waste-waters can be achieved by chemical as well as biological
means. However, the biological pretreatment offers better package over the chemical means by
being safe and economical. The biological pre-treatment can be accomplished by using a blend of
microorganisms able to withstand such harsh alkaline conditions. In the present work, for the
proper pre-treatment of alkaline waste-waters, the blend of B. alkalophilus and B. sp. are capable
of lowering the pH from 9-10.5 to pH of 5.26-8.26 after 5 days’ incubation in the presence of
carbohydrates (sucrose and glucose) (Kumar et al., 2011).
20
3 MATERIALS AND METHODS
The experimental works were conducted at Metahara Research and Development Center,
Ethiopian Public Health Institute (EPHI) and Addis Ababa Institute of Technology(AAiT)
Laboratories.
3.1 Materials and reagents
Beseka lake water was used as raw material. The reagents used in this research were hydrogen
peroxide (30%), H2O2, Phenolphthalein indicator solution, Potassium chromate indicator solution,
silver nitrate solution (0.025N), sodium chloride solution (0.025N), sodium hydroxide solution
(0.25N), sulfuric acid solution (0.02N), methyl orange indicator solution, Sodium Carbonate
(anhydrous) and basal agar medium. The apparatus and equipment’s which will be used for this
research are conductivity meter, analytical balance, standard laboratory titrimetric equipment,
including 1 mL or 5 mL micro burette with 0.01 mL gradations, and 25 mL burette, beakers,
pipettes (volumetric), volumetric flasks (1000mL, 500ml, 200mL), UV-Visible
Spectrophotometer, biological safety cabinet, autoclave and incubator.
3.2 Methods
3.2.1 Water Sampling method
The water samples were collected to analyze the selected physic-chemical properties of Beseka
Lake water. Accordingly, water samples were collected from Lake Beseka, Metahara, Fentalle
Woreda, Oromia region by systematic random sampling method from the surface at different sites
of Lake using clean poly ethylene plastic bottles (0.5L). Before taking the sample; the bottles first
were rinsed by sample water. The sample bottles then were labeled with an identification number.
Water sample used for isolation was collected by sterilized glass bottle. All water samples were
collected on the same day (morning) and transported to Metahara Research and Development
Center laboratories in ice box at 4°C before being prepared for analysis.
3.2.2 Sample Preparation and analysis
The alkalinity and chloride contents of collected samples were analyzed by titration while fluoride
was analyzed by UV-Visible Spectrophotometer method.
21
The water pH and electrical conductivity (EC) were measured by pH meter and EC meters
respectively while salinity was determined by TDS dried at 180°C. pH, alkalinity, EC and TDS
were done with three replicates while chloride and fluoride with two replicates. The water
sampling and analysis were done as per the APHA standards (APHA,1999) and described as
follows:
I. pH
The pH value of water sample was determined by electrometric method according to APHA-4500-
H+ B. In this procedure, in a clear dry small beaker 50ml water sample was taken and stirred well.
The electrode was then placed in well mixed water sample and checked for the reading in pH
meter. Finally, the electrode was waited until stable reading appeared and recorded.
II. Electrical conductivity (EC)
Electrical conductivity of water was measured by Electrical conductivity (EC) meter (APHA2520
B). In this procedure, first the meter was calibrated by immersing into standard potassium chloride
solution which has conductivity of 1413µS/cm. Then, the electrode was placed in 50ml water
sample and records the conductivity of water with the corresponding temperature.
III. Salinity (TDS)
The well-mixed water sample was filtered under vacuum pump through Gooch Crucible filter.
100mL of filtered water sample was then transferred in a weighed evaporating dish and then
evaporated to dryness on steam bath. The evaporated sample was dried to constant weight in an
oven either at 103-105°C or 179-181ºC. The dried sample was cooled in a desiccator and then
weighed. The drying procedure was repeated until constant weigh was obtained. The result will be
then analyzed as follows (APHA2540 C):
𝑇𝑜𝑡𝑎𝑙 𝑓𝑖𝑙𝑡𝑒𝑟𝑎𝑏𝑙𝑒 𝑟𝑒𝑠𝑖𝑑𝑢𝑒 (𝑚𝑔/𝐿) 𝑎𝑡 180°𝐶 = (𝐴 –𝐵) 𝑥 106
𝐶 ……………………………… (3.1)
Where: A = weight of dried residue + dish, B = weight of dish, C = mL of filtrate used, 106 =
conversion factor.
22
IV. Alkalinity test
Alkalinity of water was determined by titration following APHA2320-B standard. In this
procedure, 50mL of unfiltered water sample was taken in a conical flask and 2-3 drops of methyl
orange indicator solution was added to the flask. The sample was then titrated with 0.02 N H2SO4,
constantly swirling the flask content above a white surface until just after the colour of the flask
content change from yellow to red.
V. Chloride (Cl -) test
50 mL of water sample was poured into a white porcelain container. The end point of the silver
nitrate for titration of chloride was indicated by potassium chromate. The method number used
was APHA4500- Cl - B.
VI. Fluoride (F-) test
50ml of water sample was used to analyze fluoride ion. The concentration of fluoride in water was
determined using UV-Visible Spectrophotometer (APHA4500- F – D).
3.3 Media Composition and Isolation of Bacteria
3.3.1 Media Composition
The isolation of bacterial was done on basal media. The media contained ingredients were given
in the table 3.1as g/l of distilled water.
Table 3.1: media composition (Dhundale, 2012; Joshi & Kanekar, 2008)
Ingredient composition (g/l)
Glucose 10
Yeast extract 5
Peptone 5
KH2PO4 1
MgSO4.7H2O 0.2
Na2CO3 (anhydrous) 10
Agar (for solid medium) 20
23
3.3.2 Isolation of Bacteria
Sample was collected from Beseka Lake, Metahara, Oromia region, in order to isolate potent
microbes for biological treatment. Isolation of bacteria was done by the streak plate method. In
this method, first the 1ml of sample water was transferred in 9ml of sterilized distilled water and
mixed by vortex shaker for 20 seconds. The suspension was then diluted up to 10-6 dilutions. From
each dilution, 0.1 ml of the diluted sample were then plated onto sterilized surface plate of nutrient
agar. Then, the plate was streaked following quadrant streak method. Finally, the plate was
incubated at 35ºC for 3days. Then, the pure colonies were distinguished by size and color.
Individual bacterial isolates were further sub cultured on the basal agar medium in order to obtain
pure culture. Out of these the best grown isolate on basal agar was selected on the basis of their
growth and, maintained at 4°C in refrigerator for beseka lake water treatment and further studies
(Kumar et al., 2007; Demissie, 2014).
3.4 Experimental procedures
3.4.1 Inoculum preparation
Frozen cultures of Bacillus alkalophilus kindly given by Ethiopian Biodiversity Institute and the
isolated bacteria from Lake Beseka (good growth on basal agar medium) stored in vial and agar
slant respectively were inoculated separately on sterilized a basal broth of flask composed of 10g
glucose, 5g yeast extract, 5g peptone, 1g KH2PO4, 0.2g MgSO4.7H2O and 10g Na2CO3 at aseptic
conditions Then, the inoculated broth of flasks were covered with cotton and incubated on rotary
shaker at 35°C, 150rmp and 72 h(Sivaprakasam et al, 2008). Finally, the grown culture suspensions
were used as sources of inoculum for beseka lake water treatment experiments.
3.4.2 Viable Cell Count
The number of living cells was counted by spread plate methods. Doing spread plate by making
serial dilution from 10-1 to 10-6 then 0.1 mL of bacterial suspension broth was spread over nutrient
agar plates. Plates were incubated in incubator at 35°C for 48hours. After 48hours of incubation,
colonies were counted. The calculation of the number of cells per mL was described as follow:
𝑛𝑢𝑏𝑚𝑒𝑟 𝑜𝑓 𝑐𝑒𝑙𝑙𝑠 (𝑐𝑓𝑢/𝑚𝑙) =𝑛𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑐𝑜𝑙𝑜𝑛𝑖𝑒𝑠∗𝑑𝑖𝑙𝑢𝑡𝑖𝑜𝑛 𝑓𝑎𝑐𝑡𝑜𝑟
𝑣𝑜𝑙𝑢𝑚𝑒 𝑜𝑓 𝑖𝑛𝑜𝑐𝑢𝑙𝑢𝑚 (𝑚𝑙) ………….…………. (3.2)
24
3.5 Beseka lake water treatment procedures
Treatments of beseka lake water sample were done using Bacillus alkalophilus and isolated
bacteria at different inoculum dosage, pH and incubation time. The 500ml of volumetric flasks
were filled with 400ml of beseka lake water sample enriched with 1% glucose (Kumar et al., 2011),
autoclaved and 24ml, 28ml and 32 mL of inoculated broth of Bacillus alkaliphilic and isolated
bacteria were added to the flask with sample separately. Before inoculation of the inoculum, the
water samples were adjusted to the pH of the lake (pH of 8.5, 9 and 9.5). Then the flasks were
putted on rotary shaker at 35°C and 150rmp. In addition, the treatment processes were set at
24hr,48hr and 72hr to examine the effect of exposures time.
3.6 Experimental design for Beseka lake water treatment
Response surface methodology (RSM) was adopted in the design of experimental combinations.
The main advantage of RSM is to reduce the number of experimental runs needed to provide
sufficient information for statistically acceptable results. Box-Behnken Design (BBD) of response
surface methodology is based on the construction of balanced incompleted block designs and
requires at least three levels for each factor. In Box-Behnken experimental design, the level of one
of the factors is fixed at the center level while combinations of all levels of the other factors are
applied( Tekindal1 et al., 2012). Therefore, in this study Box- Behnken design (BBD) with three
levels for three factors was employed.
The first task before conducting the experiments was selection of potential parameters to be varied.
The three main factors selected in this study were dosage of inoculum, exposure time, and pH. The
levels of the selected factors were determined from reviewing several research literatures. The
factors among with their levels are described in Table 3.2. The experiment performed as a
completely randomized design with three main factors at three levels and two response variables.
The processing variables (dosage of inoculum, exposure time, and pH.) were optimized using RSM
of BBD to study their effect on the biological treatment of Beseka Lake. The responses that were
considered during the optimization of the Processing variables are total alkalinity and salinity
(TDS).
25
Table 3.2: Independent Variables and Levels Used in the BBD for the Beseka Lake water
treatment
Variable Unit Coded symbol Coded Levels
-1 0 +1
PH value - A 8.5 9.0 9.5
Incubation time hr. B 24 48 72
Inoculum size ml C 24 28 32
3.7 Data analysis
Data analysis has performed by DESIGN EXPERT ® 7 software using Response surface
methodology design method and randomize the runs. Randomization ensures that the conditions
in one run neither depend on the conditions of the previous runs nor predict the conditions in the
subsequent runs. Randomization is essential for drawing conclusions from the experiment, in
correct, unambiguous and defensible manner. Box- Behnken design (BBD) for a three factor
(dosage of inoculum exposure time, and pH) and three level combinations with a total number of
17 runs for each bacterium were used to conduct this study. Significance of the result was
performed using analysis of variance (ANOVA). Analysis of variance, ANOVA, is a statistical
decision making tool used for detecting any differences in average performances of tested
parameters(Manohar et al., 2013). It employs sum of squares and F statistics to find out relative
importance of the analyzed processing parameters, measurement errors and uncontrolled
parameters. It was used to check the adequacy of the model for the responses in the
experimentation.
26
4 RESULTS AND DISCUSSION
4.1 Chemical compositions of Lake Beseka water
The chemical compositions of beseka lake water were determined and presented in table 4.1
Table 4.1 characteristics of Lake Beseka water
Parameters unit Value
pH - 9.0
EC (electrical conductivity) µS/cm 3906
TDS (total dissolved solid) mg/l 3178
Cl- (chloride) mg/l 349.89
F-(fluoride) mg/l 16.13
HCO3- (bicarbonate ion) mg/l 1016.80
CO32- (carbonate ion) mg/l 216.0
4.1.1 pH
In this study, the pH of beseka lake water was found to be with the range of (8.5- 9.5) with average
value of 9. This values show that the lake is basic (alkaline) in nature (pH>7), and to a small extent
above the recommended range for fresh lake water (7.3–9.2) (Mali et al., 2012). The value of the
present study agrees with the former research result done by Ayenew and Legesse (2007) with
average value of pH (9.08). In the opposite, the pH values of Lake Beseka water was close to the
most irrigated lake (lake Ziway), which has a pH value of 8.4-.8.9 (Masresha et al., 2011). The pH
of the Ziway lake is within the permissible limits of (WHO,1993; EPA,2003) for drinking,
recreation, agricultural and aquatic life water use (6.5-8.5/9). Thus, the reasons for rising of pH
are due to the presence of ions (HCO3- and CO3
2-) while absence of dissolved CO2. The other
sources of alkaline pH can be the levels of hard-water minerals and release of basic industrial and
agricultural effluents. The presence of bicarbonate ions, HCO3-, in the lake is resulting from
limestone deposits react with water.
27
4.1.2 Electrical conductivity (EC)
As indicated in Table 4.1, the measured value of electrical conductivity (EC) of Beseka Lake was
between 3032 to 4780 µS/cm, with average value of 3906 µS/cm, such that the present value was
far above the WHO (WHO, 1993) guideline value prescribed for drinking purpose (1500 µS/cm)
and EPA guideline (1000 µS/cm) and for livestock and poultry watering purposes (<1000 µS/cm).
Even, the value of EC in the lake was higher than lake Ziway(479-530µS/cm) (Masresha et al.,
2011). Higher the EC, less amount of water will be available to plants, even though the soil may
appear wet. This is because plants can only transpire "pure" water as the usable plant water in the
soil solution decreases dramatically with an increase in EC (Abduro and W/michael, 2017).
Therefore, irrigation water with high EC reduces yield potential. This higher amount of EC from
beseka lake was due to the presence of higher value of dissolved salts ions in the water. The large
variations in the EC and TDS may be attributed to variation in geochemical processes and
anthropogenic activities( Mondal et al., 2008). Generally, there are anthropogenic activities like
discharge of effluents from municipals of MER, agrochemical run off, effluents discharged nearby
MER factories without treatment, and the natural processes like precipitation inputs, erosion,
weathering of crustal materials are contributors of higher values EC and TDS in Lake Beseka water
(Abduro and W/michael, 2017; Olumana and Loiskandl, 2015).
4.1.3 Total dissolved solid (TDS)
Salinity or Total Dissolved Solids (TDS) is a measure of the total ionic concentration of dissolved
minerals in water. There is a positive correlation between EC and TDS. The TDS of the Lake
Beseka (2425 to3930 mg/L which has average value of 3178 mg/l ) were greater than the Lake
Ziway which ranges from 200 to 400 mg/L (Worako, 2015). The value of TDS decreased in the
lake in comparing with the previous researches done by Ayenew & Legesse (2007)
(TDS=4936mg/l) and Olumana and Loiskandl (2015) (5000-12,000mg/l). The possible reason for
reduction of TDS was probably due to the excessively high amount of flood discharged into the
Lake from Koka dam and irrigation runoff, and microbial degradation of dissolved solid. TDS is
composed of the following principal cations (or positively charged ions): Sodium (Na+), Calcium
(Ca2+), Potassium (K+), Magnesium (Mg2+), and anions (or negatively charged ions): Chloride (Cl-
), Sulfate (SO4 2-), Carbonate (CO3
2-), Bicarbonate (HCO3-), and, to a lesser extent by Nitrate
(NO3-), Boron (B3+), Iron (Fe3+), Manganese (Mn2+) and Fluoride (F-) (Siosemarde et al., 2010).
Therefore, in lake beseka, cations like Sodium (Na+), Potassium (K+), and anions like Chloride
28
(Cl-), Sulfate (SO4 2-), Bicarbonate (HCO3
-), and Fluoride (F-) are major sources for rise of TDS (
Olumana & Loiskandl, 2015). High concentration of dissolved ions can damage the organism’s
cells and also reduced the photosynthesis activity and increases the water turbidity and water
temperature.) (Mondal et al, 2008).
4.1.4 Chloride (Cl-)
The existence of chloride and fluoride in water in excess amounts is not desirable. In the present
investigation, the concentration of Clˉ was reached to 349.89mg /l, which was in far above the
prescribed limits set by WHO (1993), 250 mg/L for drinking water, higher concentration of Cl in
drinking water causes a salty taste and has a laxative effect in people not accustomed to it (Mondal
et al, 2008). The value obtained in the present study decreased in the lake in comparing with the
previous researches done by Olumana & Loiskandl (2015) (937mg/l) and Ayenew & Legesse
(2007) (525mg/l). The decrease in measured value of Cl-could be associated with the sampling
period and a dilution effect due to overflow of Koka Dam and irrigation runoff. Among the
halogens (Cl, Br, I, F), chloride is the most abundant. Cl ion usually exists in the form of chlorine
salts (NaCl, CaCl and MgCl) and is extremely soluble in water. High chloride content in the Rift
Valley lakes is primarily contributed from the alkaline/saline soils in the drainage area and due to
the pollution from chloride rich effluents of sewage and municipal waste(Njenga, 2004).
4.1.5 Fluoride (F-)
Fluoride is an essential element for maintaining normal development of teeth and bones. A fluoride
value in the present study was recorded as 16.13mg/l, which was far apart from the recommended
limit (WHO,1993; EPA, 2003) of F- concentration for the drinking water (1.5mg/l). Concentrations
in drinking water above the permissible limit (1.5 mg/L) causes dental fluorosis. In addition,
continuous intake of 3 mg/L to 6mg/L fluoride content water for a long period may leads to skeletal
fluorosis, if these concentrations exceeded, crippling skeletal fluorosis occur(Worako, 2015).
Fluorine is a common element that does not occur in the elemental state in nature because of its
high reactivity. It is present in the soil and rock formation in the form sellaite (MgF2), fluorspar
(CaF2), cryolite (Na3AlF6) and fluorapatite [3Ca3(PO4)2Ca(F,Cl2)], and due to anthropogenic
sources (aluminum industries, phosphate industries, coal plants, cosmetics, etc.) (Mondal et al,
2008). Furthermore, the volcanic activity and weathering processes let fluorine to be introduced
29
into groundwater and thus results an increase in the levels of fluoride. The possible sources of
fluoride in Beseka Lake may be due to chemical weathering and volcanic activity
4.1.6 Alkalinity
Alkalinity of water is described as the presence of all those substances in water which can resist
the change in pH when an acid is added to water. Total alkalinity of water is due to CO32-
(carbonate ion) and HCO3-(bicarbonate ion) anions(Ali et al., 2014). The concentration of HCO3
-
and CO32- are 1016.80mg/l and 216mg/l in study area respectively. According to this study, the
concentration of HCO3- compared to CO32- is negligible. The value of total alkalinity (carbonate
and bicarbonate) obtained in the present study decreased in the lake(1232.80mg/l) in comparing
with the previous researches done by Olumana and Loiskandl (2015) (1500mg/l) and Ayenew and
Legesse (2007) (2838mg/l). The microbes are the possible reasons for neutralization of alkaline
waste water. HCO3- value in the present study higher than Lake Ziway (255mg/l) (Masresha et
al., 2011). Based on this reality the Lake Beseka water is not suitable for the drinking, irrigation,
& livestock watering purposes. The major source of bicarbonate and carbonate are the dissolution
of carbonate rocks containing calcite (CaCO3) and dolomite [CaMg(CO3)2] and the remainder
from silicate minerals like albite (2NaAlSi3O8)(Njenga, 2004; Mondal et al, 2008). The presences
of high values of alkalinity in water have leaded unpleasant taste and may be deleterious to human
health with high pH, TDS, and total hardness(Ogunbode et al., 2016; Olumuyiwa et al., 2012).
4.2 Isolation and viable cell count
4.2.1 Isolation and selection of bacteria
In the present work, three different types of colonies were observed on agar plates that were
transferred on basal agar medium. Out of which one strain named T1 showed good growth. This
isolate strain has red color and rod shape form in agar plate. This strain was selected for beseka
lake water treatment study.
4.2.2 Viable cells count for isolate (T1) and Bacillus alkalophilus
The plate spread technique were used to count the nubmer of viable cells present in a sample and
the result was tabulated in Table 4.2. As indicated in Table 4.2, the isolate have higher colonies
than B.alkalophilus. This implies that, basal medium, temperature and incubation time are good
conditions for replication of isolate than B.alkalophilus.
30
Table 4.2 Populations of isolate (T1) and Bacillus alkalophilus
Strain Dilution Number of
colonies
Plate count
B. alkalophilus 10-6 110 1.1*109 cfu/ml
Isolate (T1) 10-6 142 1.42* 109 cfu/ml
4.3 Statistical analysis of the experimental result
Effect of pH, incubation time and inoculum size on the treatments of Beseka Lake water were
studied and presented in APPENDIX A (Table A-1 and A-2). A total of 34 experimental data for
isolated and bacillus alkalophilus bacteria were collected for treatments of Beseka Lake water
sample. These data were obtained according to experimental design developed by response surface
methodology (RSM) using box Behnken design (BBD). These results are an input for further
analysis. Since the isolate (T1) have good removal efficiencies of total alkalinity and total dissolved
solid (TDS), throughout this study, only experimental observation for isolated bacteria was
discussed.
4.3.1 Statistical analysis for isolate (T1)
Usually, it is essential to confirm first whether the fitted model provides an adequate
approximation of the actual values or not. The adequacy of the model was checked by analysis of
variance (ANOVA) and some diagnostic plots. Analysis of variance (ANOVA) is employed to test
the significance of the developed models. Table 4-3 and Table 4-4 are show the summary of the
analysis of variance (ANOVA) of the two responses total alkalinity and TDS respectively for the
isolated bacteria. The F-value is measure of variation of the data about the mean. The high F-value
and a very low probability indicate that the present models are in a good prediction of the
experimental results. The p-value serves as a tool for checking the significance of each of the
coefficients. The variables with low probability levels contribute to the model, whereas the others
can be neglected and eliminated from the model. Values of Prob> F less than 0.0500 indicate that
model terms are significant. In this study A, B, C, AB, AC, BC, A2, B2 and C2 for the response of
total alkalinity and A, B, C, AC, BC, A2 and B2 for the response of TDS are significant model’s
terms.
31
Table 4.3 Analysis of variance for Total alkalinity
Source Sum of
Squares
Df Mean
Square
F Value p-value
Prob > F
Remark
Model 1.336E+005 9 14839.04 636.44 < 0.0001 Significant
A 3666.68 1 3666.68 157.26 < 0.0001
B 2984.55 1 2984.55 128.01 < 0.0001
C 1.166E+005 1 1.166E+005 4999.63 < 0.0001
AB 1651.20 1 1651.20 70.82 < 0.0001
AC 923.55 1 923.55 39.61 0.0004
BC 1509.71 1 1509.71 64.75 < 0.0001
A2 344.70 1 344.70 14.78 0.0063
B2 795.42 1 795.42 34.12 0.0006
C2 5051.46 1 5051.46 216.66 < 0.0001
Residual 163.21 7 23.32
Lack of
Fit
135.46 3 45.15 6.51 0.0510 not
significant
Pure Error 27.75 4 6.94
Cor Total 1.337E+005 16
32
Table 4.4 Analysis of variance for TDS
The model adequacy was further investigated using R2 for the two responses, which were found
to be 0.9988 and 0.9924 for total alkalinity and TDS respectively. For a good statistical model, the
R2 value should be close to one. The results ensured a satisfactory adjustment of the quadratic
model to the experimental data. It was indicated that approximately 99.88 % and 99.24 % of the
variability in the dependent variables of total alkalinity and TDS respectively could be explained
by these models. The value of R2 (correlation coefficient) for all responses is very high and close
to the one which indicates a good agreement between experimental and predicted values.
‘Predicted R-squared’ values are in reasonable agreement with the ‘Adjusted R-Squared’ values.
"Adeq Precision" measures the signal to noise ratio. A ratio of adequate precision greater than 4 is
Source Sum of
Squares
Df Mean
Square
F Value p-value
Prob > F
Remark
Model 1.860E+005 9 20668.48 101.90 < 0.0001 significant
A-pH 6160.50 1 6160.50 30.37 0.0009
B-
Incubation
time
17112.50 1 17112.50 84.37 < 0.0001
C-
Inoculum
size
1.405E+005 1 1.405E+005 692.46 < 0.0001
AB 529.00 1 529.00 2.61 0.1504
AC 2025.00 1 2025.00 9.98 0.0159
BC 6084.00 1 6084.00 30.00 0.0009
A^2 12096.67 1 12096.67 59.64 0.0001
B^2 1928.25 1 1928.25 9.51 0.0177
C^2 40.46 1 40.46 0.20 0.6686
Residual 1419.80 7 202.83
Lack of Fit 1181.00 3 393.67 6.59 0.0500 not
significant
Pure Error 238.80 4 59.70
Cor Total 1.874E+005 16
33
desirable. In case of this study the ratio for the two responses were greater than 4, which is 83.483
for total alkalinity and 43.532 for TDS. This indicates an adequate signal.
Table 4.5 Model adequacy measures for total alkalinity
Table 4.6 Model adequacy measures for TDS
Std. Dev. 14.24 R-Squared 0.9924
Mean 2192.41 Adj R-Squared 0.9827
C.V. % 0.65 Pred R-Squared 0.8972
PRESS 19269.13 Adeq Precision 43.532
The response total alkalinity equation was obtained for the Beseka Lake treatment:
Final Equation in Terms of Coded Factors:
Total alkalinity = +982.69+21.41× A −19.31 ×B −120.71 × C− 20.32 × A ×B +15.19×
A×C− 19.43 × B × C +9.05 ×A2 −13.74 ×B2 −34.64 × C2
Final Equation in Terms of Actual Factors:
Total alkalinity = +3571.46125 −740.09850 × pH +22.39044 × Incubation time +32.38794
×Inoculum size −1.69313× pH × Incubation time +7.59750× pH × Inoculum size− 0.20237
×Incubation time× Inoculum size +36.19200× pH2− 0.023862 ×Incubation time2− 2.16481×
Inoculum size2
The response TDS equation was obtained for the Beseka Lake treatment:
Final Equation in Terms of Coded Factors:
TDS = +2175.80 +27.75 × A −46.25 × B− 132.50 × C− 11.50× A × B− 22.50× A × C
−39.00 × B × C +53.60× A2 −21.40× B2 +3.10× C2
Final Equation in Terms of Actual Factors:
TDS = +16334.00000− 3442.70000× pH +21.63958× Incubation time +76.77500 ×
Inoculum size− 0.95833× pH × Incubation time −11.25000× pH × Inoculum size−
0.40625× Incubation time × Inoculum size +214.40000 × pH2− 0.037153× Incubation time2
Std. Dev. 4.83 R-Squared 0.9988
Mean 964.18 Adj R-Squared 0.9972
C.V. % 0.50 Pred R-Squared 0.9835
PRESS 2210.71 Adeq Precision 83.483
34
+0.19375 × Inoculum size2
4.3.2 Effect of Process Parameters on the treatments of beseka lake water sample
Here, direct effect, interaction effect or a comparison between any two input parameters have been
discussed and the third parameter would be on its central level. 3D surface plots were drawn by
using BBD methods to investigate the effect of all the factors on the responses. The data obtained
from the software were discussed below.
A. Direct Effect
Effect of pH
The selection of optimum pH is essential for removal of total alkalinity and TDS from wastewater.
The effect of pH on removal of total alkalinity and total dissolved solid (TDS) for isolate (T1) are
shown in figure 4.1. It is evident from figure 4.1 that increasing pH value to 9 results in increasing
removal of total alkalinity and TDS. However, increasing pH after 9 decrease removal of total
alkalinity and TDS from original value, which mean that, increasing in pH value beyond 9 has
negative effect on metabolic activity of microbes. The microbes can’t withstand the pH beyond 9.
Figure 4.1 Effect of pH on (a)Total alkalinity (b) TDS
35
Effect of incubation time
Figure 4.2 shows effects of incubation time on reductions of total alkalinity and TDS. Total
alkalinity removal at 24hr was 857.74mg/l, 868.81mg/l at 48hr and 774.76mg/l at 72hr, and TDS
removal at 24 was 2100, 2120 at 48hr and 1940 at 72hr. As shown from Figure 4.2 increasing an
incubation time have a fluctuation on total alkalinity and TDS removal. However, the removal of
total alkalinity and TDS at high level of the incubation time might be due to the fact that the
organism grew slowly and the time period for the bacteria to reach in the stationary phase was
increased. The declines in growth of microbial populations is due to either the exhaustion of
available nutrients or by the accumulation of toxic products of metabolism(Lee, 2002). Production
of this toxic matter (acid in this case) contribute for neutralization of alkalinity of water.
Figure 4.2 Effect of incubation time on (a)Total alkalinity (b) TDS
Effect of inoculum size
Removal of total alkalinity and TDS were investigated using different inoculum dosage. Figure
4.3 illustrates the effects of inoculum size on reduction of total alkalinity and TDS. As observed
from the Figure, the inoculum size had a positive effect on the reductions of total alkalinity and
TDS of water sample.
36
The removal of total alkalinity and TDS were increased with increase in the size of inoculum. As,
the inoculum level was further increased, the removal of total alkalinity and TDS were gradually
increased. However, the removal of total alkalinity and TDS at low level of the inoculum might
be due to the fact that the organism grew slowly and the time period for the bacteria to reach in the
stationary phase was increased. This is agreeing with the study done by Jain et al., (2011). This
behavior was due to the fact that, as the inoculum size increase, much amount of acid produced by
the bacteria colonies, which facilitated neutralization of alkaline water, and also the presence of
more inoculum increases degradation of TDS. The maximum reductions of total alkalinity and
TDS were found at inoculum size of 32ml.
Figure 4.3 Effect of inoculum size on (a)Total alkalinity (b) TDS
B. Interaction Effect
In this section only, the interaction effect of inoculum size and pH was plotted, while the plot of
the other interaction effects for all responses was given at APPENDIX B (Table B -1 and B -2).
The interaction effects of these variables on the biological treatments of beseka lake water were
studied by plotting 3D surface against the two independent variables. The three-dimensional
surface for the responses as a function of inoculum size and pH was shown in Figure 4-4 using the
design expert statistical software, 7.0.0 trial version.
37
(a)
(b)
Figure 4-4 Interaction effects of pH and inoculum size on reduction of (a) total alkalinity (b)
TDS
From the response plot in Figures 4-4 (a) and (b) observed that, Interaction effects of inoculum
size and pH have positive effect on the removal of total alkalinity and TDS. Removal of total
alkalinity and TDS increases as increasing inoculum size and pH. The maximum reduction of total
Design-Expert® Softw are
Total alkalinity
1085.14
774.76
X1 = A: pH
X2 = C: Inoculum size
Actual Factor
B: Incubation time = 72.00
8.50
8.75
9.00
9.25
9.50
24.00
26.00
28.00
30.00
32.00
760
840
920
1000
1080
Tot
al a
lkal
inity
A: pH
C: Inoculum size
Design-Expert® Softw are
TDS
2420
1940
X1 = A: pH
X2 = C: Inoculum size
Actual Factor
B: Incubation time = 72.00
8.50
8.75
9.00
9.25
9.50
24.00
26.00
28.00
30.00
32.00
1930
2042.5
2155
2267.5
2380
TD
S
A: pH
C: Inoculum size
38
alkalinity and TDS obtained at maximum inoculum size (32ml) and medium pH (9). This is due
that high inoculum size can be neutralize the alkaline Beseka Lake water in short time, and the
microbials activity decreases beyond pH 9. Even if the isolated bacteria had high reduction of
total alkalinity and TDS than provided one (bacillus alkalophilus), not only the interaction effects
of inoculum size and pH but also interaction effects (pH & incubation time and incubation time &
inoculum size for total alkalinity) and (incubation time &inoculum size for TDS) have the same
trained for bacillus alkalophilus.
4.4 Optimization of process factors and response variables for isolate
One of the specific objectives of the present study was to find the optimum process parameters to
get the highest reduction of total alkalinity and TDS. In order to determine the optimum processing
conditions, viewed by response surface plot, pH, incubation time, inoculum size, total alkalinity
and salinity (TDS) were considered to be in range, in range, in range, minimum and minimum
respectively. Table 4-7 shows the summary of factors/responses and goals for isolated bacteria.
Table 4-7 exhibits the desired combinations of process parameters that would provide the highest
responses.
Table 4-7: Constrains applied for optimization
Lower Upper Lower Upper
Name Goal Limit Limit Weight Weight Importance
pH is in range 8.5 9.5 1 1 3
Incubation
time
is in range 24 72 1 1 3
Inoculum
size
is in range 24 32 1 1 3
Total
alkalinity
minimize 774.76 1085.14 1 1 3
TDS minimize 1940 2420 1 1 3
Numerical optimization was used to find the optimum value of the process parameters and for the
responses. Since the developed model was capable of predicting the maximum value, therefore,
from Figure 4-5 the ramp plot shows that the highest reduction of total alkalinity and TDS were
39
found at a pH of 8.99, incubation time of 71.99 and inoculation size of 32ml and which were
resulted 774.669mg/l in total alkalinity and 1939.92mg/l in TDS.
Figure 4-5: Ramp display of desirability
4. 5 Comparison of alkalinity and TDS removal from wastewater
The optimized value of biological beseka lake water treatment is indicated in the Table 4.5. Table
4.5 illustrated that the highest removal of total alkalinity and TDS were found to be 774.669mg/l
and 1939.92mg/l respectively. This means that 37% of Alkalinity and 20% of TDS were removed
from original value of 1229.31mg/l for alkalinity and 2422mg/l for TDS. In the previous work as
Reported in the literature, alkalinity removal percentage of 57.4% was found by Verma (2012)
using pulp and paper wastewater effluent. Compared to my study, this value was high. This is
due to the absence of reasonable nutrient or organic matter in beseka lake that enhances
populations of microorganism. The other reason is may be due to experimental procedures. 22%
of TDS removal was obtained by Jaint (2011) from chlor-alkali industry wastewaters as reported
in literature and this value is high compared to my work. This is due to using glucose and peptone
as carbon and nitrogen sources in addition to industry waste. The other reason is may be due to the
absence of enough transfer of oxygen in flask(reactor). However, treatment with acid (nitric acid,
hydrochloric acid and sulfuric acid) increase the TDS content (Jaint (2011).
pH = 8.99
8.50 9.50
Incubation time = 71.99
24.00 72.00
Inoculum size = 32.00
24.00 32.00
Total alkalinity = 774.669
774.76 1085.14
TDS = 1939.92
1940 2420
Desirability = 1.000
40
5. CONCLUSION AND RECOMMENDATION
5.1 Conclusion
This study result indicated that the use of Lake Besaka for irrigation and drinking purpose is
difficult. The lake water has high TDS, alkalinity and fluoride. It is possible to say that the quality
of such lake leading in damaging water resources and soil quality of the region groundwater
quality. This is a reason for the abstains of investors from investing in Metahara area. This research
was designed to utilize microorganism for treatment of beseka lake water. The Present study
concluded that isolate (T1) and Bacillus alkalophilus are able to neutralize alkaline beseka lake
water with concomitant reduction in TDS, in the presence of glucose as carbon source, and the
isolated bacteria (T1) have good neutralizing ability of the lake than Bacillus alkalophilus.
It is possible to conclude from the results obtained that beseka lake water treatment using the
isolate (T1) and Bacillus alkalophilus are influenced by the pH, incubation time and dosage of the
inoculum. Design Expert software was used to develop design of experiment and analyzed the
results. Box Behnken design in Response surface method was used to treat beseka lake water. And
for this particular study the quadratic model was the best fit model for all responses as the p-value
of this model was smaller than the other models and had the highest p-value for Lack of Fit Tests.
Among the three parameters studied, ANOVA showed that, inoculum size was found to be the
most important parameter influencing the treatment of the lake water followed by pH, while
incubation time had the least effect relative to the two factors. As the inoculum size increased from
24ml to 32ml the neutralizing efficiencies of bacteria also increased.
The optimal treatment of the lake for isolated (T1) and Bacillus alkalophilus were resulted at a pH
of 8.99, incubation time of 71.99hr and inoculum size of 32 ml. And the highest removal of total
alkalinity and TDS value were 774.669mg/l and 1939.92mg/l for isolate and 779.631mg/l and
1944.97mg/l for Bacillus alkalophilus respectively. The data revealed that, the values of the total
alkalinity and TDS doesn’t meet National Environmental Quality Standard (EEPA, 2003) and
WHO guidelines (1993). However, compared to untreated, treated values of those parameters
through biological treatment method reduces their effects on downstream irrigation system.
41
5.2 Recommendation
In this study, biological treatment of beseka lake was done under different pH, incubation time and
inoculum size. From the knowledge and insight gained during the course of this work, the
following future work is recommended in the treatment of beseka lake water:
The literature and the present study demonstrated the success to employ microbes for
alkaline wastewater treatment; however, further investigation is recommended for
biological treatments of Beseka Lake water.
Even the lake water was treated biologically, integration of physical and chemical with
biological treatment could be needed for enhancement of treatment efficiency.
Due to limitation of time only isolation of bacteria was done. For future work,
investigations on morphological and biochemical tests of the isolate is also recommended.
In this study, pH, incubation time and inoculum size considered as operating parameters. One can also investigate the effects of salt concentration as other operating parameters for
future work.
It is recommended to consider saline-alkaline beseka lake as serious problem and taking
remediation technique by government and supporters of government.
In Ethiopia, there are a factory that generate alkaline wastewaters. Those alkaline
wastewaters are either chemically treated or discharged to environment without treatment.
It is better to treat those wastes with biological method, which is environmental friendly
and cost-effective method than that of chemical one.
42
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47
APPENDIX
Appendix A: Experimental Results for the isolated and bacillus alkalophilus bacteria
Table A1: Experimental observation for isolated bacteria
Standard
order Run
Factor 1
A:pH
Factor 2
B:
Incubation
Time ( hr)
Factor 3
tC:Inoculum
Size (ml)
Response 1
Total
alkalinity
(mg/l)
Response 2
TDS (mg/l)
1 2 8.50 24.00 28.00 951.3 2230
2 7 9.50 24.00 28.00 1037.83 2289
3 13 8.50 72.00 28.00 958.8 2150
4 17 9.50 72.00 28.00 964.06 2163
5 4 8.50 48.00 24.00 1075.79 2300
6 15 9.50 48.00 24.00 1085.14 2420
7 9 8.50 48.00 32.00 798.68 2090
8 16 9.50 48.00 32.00 868.81 2120
9 5 9.00 24.00 24.00 1055.01 2297
10 6 9.00 72.00 24.00 1049.74 2293
11 10 9.00 24.00 32.00 857.74 2100
12 3 9.00 72.00 32.00 774.76 1940
13 12 9.00 48.00 28.00 981 2166
14 11 9.00 48.00 28.00 983.23 2180
15 8 9.00 48.00 28.00 979.07 2169
16 14 9.00 48.00 28.00 984.97 2181
17 1 9.00 48.00 28.00 985.2 2183
48
Table A2: Experimental observation for Bacillus alkalophilus
Standard
order Run
Factor 1
A:pH
Factor 2
B:Incubation
time (hr)
Factor 3
tC:Inoculum
size
(ml)
Response 1
Total alkalinity
(mg/l)
Response 2
TDS (mg/l)
1 10 8.50 24.00 28.00 955.3 2235
2 13 9.50 24.00 28.00 1042.74 2294
3 17 8.50 72.00 28.00 963.8 2155
4 16 9.50 72.00 28.00 969.06 2168
5 1 8.50 48.00 24.00 1080.65 2305
6 4 9.50 48.00 24.00 1090.1 2425
7 11 8.50 48.00 32.00 803.68 2095
8 2 9.50 48.00 32.00 873.91 2125
9 9 9.00 24.00 24.00 1060.01 2302
10 3 9.00 72.00 24.00 1054.74 2298
11 7 9.00 24.00 32.00 862.7 2105
12 14 9.00 72.00 32.00 779.76 1945
13 12 9.00 48.00 28.00 985.57 2171
14 15 9.00 48.00 28.00 988.23 2185
15 5 9.00 48.00 28.00 984.07 2174
16 8 9.00 48.00 28.00 989.84 2186
17 6 9.00 48.00 28.00 990.2 2188
49
APPENDIX B: 3D plots for the responses: for isolated and bacillus alkalophilus bacteria
(a)
Design-Expert® Softw are
Total alkalinity
1085.14
774.76
X1 = A: pH
X2 = B: Incubation time
Actual Factor
C: Inoculum size = 28.00
8.50
8.75
9.00
9.25
9.50
24.00
36.00
48.00
60.00
72.00
949
971.75
994.5
1017.25
1040
Tot
al a
lkalin
ity
A: pH B: Incubation time
Design-Expert® Softw are
Total alkalinity
1085.14
774.76
X1 = B: Incubation time
X2 = C: Inoculum size
Actual Factor
A: pH = 9.00
24.00
36.00
48.00
60.00
72.00
24.00
26.00
28.00
30.00
32.00
770
845
920
995
1070
Tot
al a
lkalin
ity
B: Incubation time C: Inoculum size
50
(b)
Figure B1: 3D plots for isolated bacteria (a) Total alkalinity (b) TDS
Design-Expert® Softw are
TDS
2420
1940
X1 = B: Incubation time
X2 = C: Inoculum size
Actual Factor
A: pH = 9.00
24.00
36.00
48.00
60.00
72.00
24.00
26.00
28.00
30.00
32.00
1930
2027.5
2125
2222.5
2320
T
DS
B: Incubation time C: Inoculum size
51
Design-Expert® Softw are
Total alkalinity
1090.1
779.76
X1 = A: pH
X2 = B: Incubation time
Actual Factor
C: Inoculation size = 28.00
8.50
8.75
9.00
9.25
9.50
24.00
36.00
48.00
60.00
72.00
954
976.5
999
1021.5
1044
T
ota
l alk
alin
ity
A: pH B: Incubation time
Design-Expert® Softw are
Total alkalinity
1090.1
779.76
X1 = A: pH
X2 = C: Inoculation size
Actual Factor
B: Incubation time = 48.00
8.50
8.75
9.00
9.25
9.50
24.00
26.00
28.00
30.00
32.00
800
875
950
1025
1100
Tot
al a
lkal
inity
A: pH C: Inoculation size
52
(a)
(b)
Figure B2: 3D plots for Bacillus alkalophilus bacteria (a) Total alkalinity (b) TDS
Design-Expert® Softw are
Total alkalinity
1090.1
779.76
X1 = B: Incubation time
X2 = C: Inoculation size
Actual Factor
A: pH = 9.00
24.00
36.00
48.00
60.00
72.00
24.00
26.00
28.00
30.00
32.00
770
847.5
925
1002.5
1080
Tot
al a
lkalin
ity
B: Incubation time C: Inoculation size
Design-Expert® Softw are
TDS
2425
1945
X1 = B: Incubation time
X2 = C: Inoculation size
Actual Factor
A: pH = 9.00
24.00
36.00
48.00
60.00
72.00
24.00
26.00
28.00
30.00
32.00
1940
2035
2130
2225
2320
TD
S
B: Incubation time C: Inoculation size
Design-Expert® Softw are
TDS
2425
1945
X1 = A: pH
X2 = C: Inoculation size
Actual Factor
B: Incubation time = 48.00
8.50
8.75
9.00
9.25
9.50
24.00
26.00
28.00
30.00
32.00
2050
2145
2240
2335
2430
TD
S
A: pH C: Inoculation size
53
APPENDIX C: Figures Taken during Laboratory Work
Figure C1: water sampling from beseka lake (a), media(b)Autoclave (c)
Figure C2: serial dilution for isolation (a)Serial dilution for counting (b), sterilization (c)
a b c
a b c
54
Figure C3: viable cell (a), pH meter (b) incubation (c)
Figure C4: Slant culture (a) Broth culture (b), untreated water before inoculation (c)
a b c
a b c
55
Figure C5: Untreated water after inoculation (a) treated water (b), Biological safety cabinet(c)
Figure C6: Isolate (a) broth culture (b) spread plating(c)
a b c
a b c